Anticancer Effect Enhancer

ABSTRACT

An object of the present invention is to provide an enhancing agent for effect of anticancer agent for achieving an excellent therapeutic effect on cancer. The enhancing agent for effect of anticancer agent according to the present invention which is a solving means therefor is characterized in that a nitric oxide donor is an effective ingredient. In accordance with the present invention, an excellent therapeutic effect is able to be achieved on non-small cell lung cancer which is still in such a state that no effective therapeutic method has been established yet for a progressive cancer which is not operable and is one of cancers where chemotherapy is most difficult to apply.

TECHNICAL FIELD

The present invention relates to an enhancing agent for effect ofanticancer agent for achieving an excellent therapeutic effect oncancer.

BACKGROUND ART

It has been well known that, as a result of vigorous research anddevelopment of anticancer agents for many years, various kinds ofanticancer agents are now being used in chemotherapy to various types ofcancer and therapeutic effect is achieved thereby. However, it has beenalso known that there is no anticancer agent which is effective on alltypes of cancer and there is a limitation for the therapeutic effect ofanticancer agent on cancer.

As one of the causes for the limitation in the therapeutic effect oncancer by anticancer agent, it has been reported that hypoxic conditioninside the solid cancer participates in resistance to its therapy. Forexample, in Non-Patent Document 1 (Matthews N E, Adams M A, Maxwell L R,Gofton T E, Graham C H. Nitric oxide-mediated regulation ofchemosensitivity in cancer cells. J. Natl. Cancer Inst., 2001; 93:1879-1885), it is shown that, in experiments where several kinds ofcancer cell lines are actually used, hypoxic condition promotes theresistance of cancer cells to anticancer agent. In this document, it ismade clear that, when an anticancer agent is mixed with culture mediumof cancer cell lines, the surviving rate is as high as two-fold or moreeven if the cancer cells are directly exposed to the anticancer agent ascompared with the case where they are exposed in normoxic conditionprovided that they are exposed in hypoxic condition. It is also madeclear that hypoxic condition suppresses an endogenous production ofnitric oxide (NO) whereby it enhances the resistance of the cancer cellsto anticancer agent and that administration of an exogenous NO donorimproves the resistance to anticancer agent by hypoxic condition.Besides that, in Non-Patent Document 2 (Jordan B F, Misson P D, DemeureR, Baudelet C, Beghein N, Gallez B. Changes In tumoroxygenation/perfusion induced by the NO donor, Isosorbide dinitrate, Incomparison with carbogen: monitoring by EPR and MRI. Int. J. RadiationOncology Biol. Phys., 2000; 48: 565-570), it is suggested thatisosorbide dinitrate which is a NO donor improves oxygen pressure insidethe cancer due to an increase in blood flow. In Non-Patent Document 3(Liang B C. Effects of hypoxia on drug resistance phenotype and genotypein human glioma cell lines. J. Neurooncol., 1996; 29: 149-155), it ismade clear that, when glioma cell line is placed in hypoxic condition,it shows resistance to anticancer agent. In Non-Patent Document 4 (SannaK, Rofstad E K. Hypoxia-induced resistance to doxorubicin andmethotrexate in human melanoma cell lines in vitro. Int. J. Cancer,1994; 58: 258-262), it is made clear that, when human melanoma cell lineis placed in hypoxic condition, it shows resistance to anticancer agent.Thus, according to those reports, even when anticancer agent is merelydistributed in large quantities in cancer cells, death of cancer cellsis not accelerated unless hypoxic condition in tumor tissues isimproved. Non-Patent Document 1 and Non-Patent Document 2 suggest that aNO donor is able to effectively function in improvement of hypoxiccondition inside the solid cancer.

However, in any of the reports, it is not proved that NO actuallyenhances the therapeutic effect of anticancer agent on cancer in humanclinical medicine. Rather, there are many reports which suggest that NOhas an action of increasing the tumor size and promoting its progress.For example, in Non-Patent Document 5 (Gallo O, Masini E, Morbidelli L,Franchi A, et al. Role of nitric oxide in angiogenesis in head and neckcancer. J. Natl. Cancer Inst., 1998; 90: 587-596), a NO synthase (NOS)participating in NO production in tissue preparation of human head andneck cancer is investigated and it is shown that, in advanced cancercases accompanied by metastasis to lymph node, expressed amount of NOSis high and blood vessel density in lymph node is high in a group wheremetastasis to lymph node is positive. In this document, an investigationusing cornea of rabbit is also conducted for tumor angiogenetic effectof NO on cancer tissues prepared from human head and neck cancer cases.According to that, it is suggested that, in a group to which L-NAMEwhich is a NOS inhibitor is administered, tumor angiogenesis issignificantly little and progress of cancer is suppressed as comparedwith a control group and it is shown that NO has a promoting action forincrease and progress of cancer tissues via tumor angiogenesis. InNon-Patent Document 6 (Edwards P, Cendab J C, Topping D B, Moldawer L L,Mackay S, Copeland E M, Lind D S. Tumor cell nitric oxide inhibits cellgrowth in vitro, but stimulates tumorigenesis and experimental lungmetastasis in vivo. J. Surg. Res., 1996; 63: 49-52), it is shown that,when an experiment using cultured cells where NO production is promotedby stimulation of LPS/IFN-γ using EMT-6 cells (breast cancer cell lineof mice) is conducted, growth of tumor cells is suppressed while, whenthe same cells are transplanted to BALB/c mouse and stimulated byLPS/IFN-γ, tumor tissues and lung metastasis increased to an extent oftwo-fold as compared with a control group and that experimental resultsare entirely opposite between an experiment using cultured cells (invitro experiment) and an animal experiment (in vivo experiment). In theNon-Patent Document 7 (Ambs S, Merriam W G, Ogunfusika M O, Bennett W P,Ishibe N, et al. p53 and vascular endothelial growth factor regulatetumor growth of NOS2-expressing human carcinoma cells. Nature Med.,1998; 4: 1371-1376), an influence of NO on angiogenesis and cancerprogress is investigated in an animal experiment where a human cancercell line into which NOS gene is introduced so as to conduct a NOsynthesis in a constant manner is transplanted to nude mice having nothymus. Here, relation to the presence/absence of an activity of cancersuppressive gene p53 is investigated and it is shown that, in tumortissues where cancer cell line having a p53 activity (wild type p53) istransplanted to nude mice, an endogenous NO suppresses the growth oftumor cells while, in tumor tissues where cancer cell line having poorp53 activity (mutant p53) is transplanted to nude mice, the endogenousNO promotes VEGF expression and angiogenesis, and promotes the growth oftumor cells. In Non-Patent Document 8 (D C Jenkins, I G Charles, L LThomsen, D W Moss, L S Holmes, S A Baylis, P Rhodes, K Westmore, P CEmson, S Moncada. Roles of Nitric Oxide in Tumor Growth. Proc. Natl.Acad. Sci. USA, 1995; 92: 4392-4396), it is shown that, inhuman breastcancer and cancer in a gynecologic field, NO production and cancergrowth show a positive correlation. In Non-Patent Document 9 (Lala P K,Chakraborty C. Role of nitric oxide in carcinogenesis and tumourprogression. Lancet Oncol., 2001; 2: 149-156), it is shown that NOS of aderived type participates in mutation of p53 of tumor of colon, lung andthroat and that NO stimulates the growth of tumor via activation ofcyclooxygenase-2 (COX-2). Thus, according to those reports, it isstrongly suggested that, in establishing a therapy of cancer byanticancer agent in human clinical medicine, the use of NO has apossibility of causing adverse effects to patients such as increase orprogress of cancer.

As mentioned above, with regard to an action of NO to cancer, each ofthe reports mentioning it participates in the direction of suppressingthe cancer and the reports mentioning it participates in the directionof worsening the cancer are present in large numbers being supported byscientific proof and a scientific evaluation therefor is in a chaoticstate. Moreover, as shown in Non-Patent Document 6, it is never rarethat the experimental results are entirely contrary between anexperiment using cultured cells and an animal experiment. In view ofsuch circumstances, in Non-Patent Document 1 for example, it is shownthat resistance of cancer cell line to anticancer agent induced byhypoxic condition is improved by a NO donor in an experiment usingcultured cells and, although such a finding will be surely valuable forshowing one direction for future research, it is not possible toconclude from such a finding that a NO donor has an action of shrinkingthe cancer tissues or an action of enhancing the effect of anticanceragent. So, it is all the more that, in human clinical medicine, aconclusion that a NO donor enhances the effect of anticancer agent isunable to be done and should not be done.

In Patent Document 1 (JP 2004-508279 A), there is proposed a method ofadministering a NO donor as a potassium channel activator as a methodfor a selective transfer of anticancer agent to cancer wherepermeability of blood vessel for sending blood to cancer cells toanticancer agent is enhanced. However, issue of a jumped conclusion thata NO donor enhances the effect of anticancer agent only by appealing anincrease in transition of an anticancer agent to tumor tissues by anincrease in permeability through blood vessel without actualconfirmation of enhancement of NO donor in the action of anticanceragent to cancer tissues even in animal experiment is not convincing atall under the present status where scientific evaluation for the actionof NO to cancer is in chaos as mentioned above. Patent Document 1 is farbehind the conclusion that a NO donor results in enhancement of effectof anticancer agent in actual human clinical medicine. In fact, withregard to data showing that improvement in therapeutic effect for cancerwas able to be achieved by that method, no data is available even foranimal experiment in Patent Document 1.

Therefore, an object of the present invention is to provide an enhancingagent for effect of anticancer agent for achieving an excellenttherapeutic effect on cancer.

DISCLOSURE OF THE INVENTION

Under the circumstance where, even in animal experiments, no conclusionis issued what effect is available by administration of a NO donor tocancer tissues, the present inventors have at this time found that a NOdonor dramatically improves the therapeutic effect of an anticanceragent on non-small cell lung cancer which is believed to be one of themost difficult cancers for chemotherapy. One of the backgrounds thereforis a retrospective clinical research in patients with either anginapectoris or old myocardial infarction treated with a NO donor. That isbecause there is a ground of a data analysis where response rate bychemotherapy is quite significantly higher in a group suffering fromadvanced non-small cell lung cancer and receiving a chemotherapy (MVPtherapy) (group where NO is used together; age: 67±8; male ratio: 80%)than a group using no NO donor and receiving the same MVP therapy(control group; age: 65±9; male ratio: 76%) where odds ratio=30.6, 95%CI 3.5-270.4, p<0.0001, Chi-square test. In the data, response rate ofthe group where NO was used together was 90% (CR: 20%, 2/10; PR: 70%,7/10; NC: 10%, 1/10; PD: 0%, 0/10) while response rate of the controlgroup was 23% (CR: 0%, 0/44; PR: 23%, 10/44; NC: 50%, 22/44; PD: 27%,12/44) (With regard to the method for judgment, refer to the Exampleswhich will be mentioned later).

The present invention has been achieved on the basis of theabove-mentioned finding, and according to a first aspect and feature ofthe present invention, there is provided an enhancing agent for effectof anticancer agent which is characterized in that a NO donor is aneffective ingredient.

According to a second aspect and feature of the present invention, inaddition to the first feature, the cancer which is an object for thetreatment is solid cancer.

According to a third aspect and feature of the present invention, inaddition to the second feature, the solid cancer is non-small cell lungcancer.

According to a fourth aspect and feature of the present invention, inaddition to the first feature, the NO donor is an organic nitratecompound.

In accordance with the present invention, it is possible to provide anenhancing agent for effect of anticancer agent for achieving anexcellent therapeutic effect on cancer.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a drawing which shows survival rate curves in patients withadvanced non-small cell lung cancer treated with NO donors {NO(+)}(solid line) and without NO donors {NO(−)} (dotted line) duringchemotherapy. The P value was calculated by the log-rank test.

FIG. 2 shows the tumor volume growth curves in lung cancer model (MurineLewis lung carcinoma; LLC cells) in C57BL6 mice (Control group, n=6; C,NO donor group, n=6; N, Chemotherapy group, n=6; CTX, and NO pluschemotherapy group, n=6; N+CTX). Tumor volume was calculated as0.5×{tumor length (mm)}×{tumor width(mm)}². The P value was calculatedby student t-test.

FIG. 3 shows the tumor volume growth curves in colon cancer model(Murine colon cancer cell line; colon 26 cells) in BALB/c mice (Controlgroup, n=6; C, NO donor group, n=6; N, Chemotherapy group, n=6; CTX, andNO plus chemotherapy group, n=6; N+CTX). Tumor volume was calculated as0.5×{tumor length (mm)}×{tumor width (mm)}². The P value was calculatedby student t-test.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, a nitric oxide (NO) donor means an agent whichis able to release NO under a physiological condition. For example,organic nitrate compounds which are nitric esters of monohydric orpolyhydric alcohol are included in this category. Their representativeexamples are nitroglycerin (NTG), pentaerythrityl tetranitrate (PETN),isosorbide dinitrate (ISDN) and isosorbide mononitrate (ISMN).

There is no particular limitation for an anticancer agent which is usedin chemotherapy where effect is enhanced by the concomitant use of a NOdonor and its examples are antimetabolites exemplified by5-fluorouracil, methotrexate, doxifluridine, tegafur, cytarabine andgemcitabine; alkylating agents exemplified by cyclophosphamide,ifosfamide, thiotepa, carboquone and nimustine hydrochloride; anticancerantibiotics exemplified by mitomycin, doxorubicin hydrochloride,amurubicin hydrochloride, pirarubicin hydrochloride, epirubicinhydrochloride, aclarubicin hydrochloride, mitoxantrone hydrochloride,bleomycin hydrochloride and peplomycin sulfate; microtubule-actingagents exemplified by docetaxel, paclitaxel, vincristine, vindesine andvinorelbine; platinum-based agents exemplified by cisplatin, carboplatinand nedaplatin; topoisomerase inhibitors exemplified by irinotecan andnogitecan hydrochloride; and alkaloidal anticancer agents exemplified byetoposide.

With regard to a NO donor, an example is that its administration isstarted to a patient who is subjected to a standard chemotherapy from 5to 2 days before the start of the chemotherapy (preferably, 3 daysbefore), the administration is continued during the period ofchemotherapy and, at the stage where chemotherapy is finished, theadministration is stopped. Although there is no particular limitationfor the dosage form of the NO donor, it is preferred to be an oralpreparation or a subcutaneous preparation where control of concentrationin blood is easy when risk of expression of resistance is taken intoconsideration. The dose may be in accordance with a dose in thetreatment of angina pectoris which is an inherent use of a NO donor. Tobe more specific, when nitroglycerin is used, administration may be in adose of 1 to 50 mg/day (being divided into two administrations a day) inthe case of oral administration by means of tablets or the like and 1 to50 mg/day (applied once daily) in the case of transdermallyadministration by means of plaster or the like. When isosorbidedinitrate is used, administration may be in a dose of 10 to 100 mg/day(being divided into two administrations a day) in the case of oraladministration and 10 to 100 mg/day (applied once daily) in the case oftransdermally administration. Incidentally, the above description doesnot deny the administration of a NO donor in a form of a combinedpreparation with an anticancer agent.

There is no particular limitation for the cancer which is an object ofthe treatment in the present invention but any cancer may be applied sofar as it is a cancer which is able to be an object of chemotherapy.Specific examples thereof are solid cancer such as head and neck cancer,stomach cancer, colon cancer, rectum cancer, hepatic cancer, gall andbile duct cancer, pancreatic cancer, lung cancer, breast cancer, bladdercancer, prostate cancer and uterine neck cancer, and blood cancer suchas malignant lymphoma and leukemia.

EXAMPLES

The present invention shall be described as following. Any limitationsin interpretation will not arise according to following documents.

Example 1 A Study of the Effect of Concomitant Use of NO Donors withAnticancer Agents on Response to Chemotherapy in Patients with AdvancedNon-Small Cell Lung Cancer in a Prospective Randomised Controlled Trial

Characteristics of the Subjects

Sixty five patients with inoperable advanced non-small cell lung cancer(NSCLC) fit the following five criteria and were recruited in thisstudy: (a) stage IIIB or stage IV; (b) no prior chemotherapy orradiotherapy; (c) good performance status: a performance status of 0-2according to the Eastern Cooperative Oncology Group (ECOG) scale; (d)without brain metastasis; (e) adequate renal function, hepatic function,hematological function and cardiac function.

Of the 65 patients with advanced NSCLC, 31 patients had squamous cellcarcinoma (19 patients in stage IIIB and 12 patients in stage IV), 29patients had adenocarcinoma (9 patients in stage IIIB and 20 patients instage IV), 5 patients had large cell carcinoma (4 patients in stage IIIBand 1 patients in stage IV), and all patients were randomly assigned toreceive anticancer agents with or without NO donors during chemotherapyin a prospective cohort study.

Thirty two of the 65 patients with advanced NSCLC were treated withchemotherapy with a NO donor (transdermally applied nitroglycerin,Nitroderm TTS®, 25 mg/body daily, or orally administered isosorbidedinitrate, Nitrol R®, 40 mg/body daily divided into two administrations,between 3 days before the start of each course of chemotherapy and thefinish of administration of anticancer agents). In group treated with aNO donor, 21 of 32 patients were treated with MVP, cisplatin (CDDP) plusvinorelbine (VNR) plus mitomycin (CDDP 80 mg/m² day 1, VNR 25 mg/m² day1 and 8, mitomycin 8 mg/m² day 1, in every 21 days), 6 of 32 patientswere treated with CDDP plus docetaxel (DOC) (CDDP 80 mg/m² day 1, DOC;75 mg/m² day 1, in every 21 days), and 5 of 32 patients were treatedwith CDDP plus VNR (CDDP 80 mg/m² day 1, VNR 25 mg/m² day 1 and 8, inevery 21 days). Another 33 of the 65 patients with advanced NSCLC weretreated with chemotherapy without a NO donor. In group treated without aNO donor, 18 of 33 patients were treated with MVP, CDDP plus VNR plusmitomycin (CDDP 80 mg/m² day 1, VNR 25 mg/m² day 1 and 8, mitomycin 8mg/m² day 1, in every 21 days), 8 of 33 patients were treated with CDDPplus DOC (CDDP 80 mg/m² day 1, DOC; 75 mg/m² day 1, in every 21 days),and 7 of 33 patients were treated with CDDP plus VNR (CDDP 80 mg/m² day1, VNR 25 mg/m² day 1 and 8, in every 21 days). Characteristics of thesubjects in arm A and arm B are shown in Table 1. TABLE 1Characteristics of patients with advanced non-small cell lung cancer.With NO mimetics Without NO mimetics Characteristics (Arm A, n = 32)(Arm B, n = 33) P value Age (year) Median 68 67 0.2615* Range 41-8348-84 Gender (No. of patients) Male 29 24 0.063 Female  3  9 PerformanceStatus (No. of patients) 0 23 23 0.8469 1  6  6 0.9529 2  3  4 0.721Brinkman Index (pack-year) Median 49 47 0.6334* Range  0-120  0-125 Celltype (No. of patients) Squamous cell 18 13 0.1737 Adenocarcinoma 10 190.0328 Large cell  4  1 0.1520 Stage (No. of patients) III B 17 150.5363 IV 15 18 Chemotherapy (No. of patients) MVP 21 18 0.362 CDDP +DOC  6  8 0.5902 CDDP + VNR  5  7 0.5616MVP = mitomycin C + vinorelbine + cisplatin,CDDP = cisplatin,DOC = docetaxel,VNR = vinorelbine.Two factors marked * was statistically assessed with the Mann-Whitney Utest.Other factors was assessed with the Chi-square test.Methods

NO donors were used between 3 days before the start of each course ofchemotherapy and the finish of administration of anticancer agents. Toassess the effects of NO donors on the chemosensitivity, we compared thetumor sizes with the chest computed tomography (CT) scan before andafter treatment with anticancer agents. The nodal staging of lung cancerwas determined using a CT scan and a Gallium-67 citrate scintigram ofthe chest. The metastasis in the brain, the abdomen and the bone wasdetermined using a CT scan of the brain and abdomen, and aTechnetium-99m scintigram of the bone. Complete response (CR) wasdefined as the disappearance of all known disease determined by twoobservations not less than two weeks apart. Partial response (PR) wasdefined as a 50% or more decrease in the total tumor size of thoselesions measured during two observations not less than four weeks apart.No change (NC) was defined as cases where a 50% decrease in the totaltumor size could not be established, and also there was not a 25% orgreater increase in the size of one or more measurable lesions.Progressive disease (PD) was defined as a 25% or greater increase in thesize of one or more measurable lesions or the appearance of new lesions.The patients were categorized as responders when they experienced eitherPR or CR. The patients with NC or PD were categorized as non-responders.Response rate to chemotherapy was calculated by the number of respondersdivided by the number of responders plus non-responders. Survival wascalculated from the date of the first day of first cycle of thechemotherapy to the date of death or a cutoff date for patients alive atthe time of closure of the data set.

The factors associated with the response to the chemotherapy wereassessed by univariate analysis (Chi-square test) and multivariateanalysis (Logistic regression analysis). Cox regression analysis wasperformed to assess the prognostic significance of the variables. Weanalyzed the survival rate with the Kaplan-Meire method, and the p-valueof survival curves between patients treated with anticancer agents andNO donors and patients treated with anticancer agents alone with theLog-rank test. Significance was accepted at p<0.05.

Explanation of Technical Term (No. 1)

ECOG Performance Status (PS): Grade ECOG 0 Fully active, able to carryon all pre-disease performance without restriction 1 Restricted inphysically strenuous activity but ambulatory and able to carry out workof a light or sedentary nature, e.g., light house work, office work 2Ambulatory and capable of all selfcare but unable to carry out any workactivities. Up and about more than 50% of waking hours 3 Capable of onlylimited selfcare, confined to bed or chair more than 50% of waking hours4 Completely disabled. Cannot carry on any selfcare. Totally confined tobed or chair 5 Dead* Am J Clin Oncol 5: 649-655, 1982.Explanation of Technical Term (No. 2)Brinkman Index: a smoking history calculated by multiplying the numberof cigarettes consumed a day (number of packages of cigarettes) bysmoking duration (years).Results

Characteristics of the subjects in responders and non-responders areshown in Table 2 and Table 3. Survival curves for the group treated withNO donors and the group treated without NO donors calculated byKaplan-Meier analysis are shown in FIG. 1. TABLE 2 Analysis of riskfactors for chemosensitivity assessed by multivariate analysis. PR + CRNC + PD Multivariate (Responder) (Non-responder) analysisCharacteristics (n = 39) (n = 26) p value Age (year) 65≧ 30 (67%) 15(33%) 0.792 65<  9 (45%) 11 (55%) Gender (No. of patients) Male 35 (66%)18 (34%) 0.438 Female  4 (33%)  8 (67%) Performance Status (No. ofpatients) 0 28 (61%) 18 (39%) 0.927 1  6 (50%)  6 (50%) 2  5 (71%)  2(29%) Brinkman Index (pack-year) 50≧ 20 (74%)  7 (26%) 0.899 50< 19(50%) 19 (50%) Cell type (No. of patients) Squamous cell 26 (84%)  5(16%) 0.011 Adenocarcinoma 10 (34%) 19 (66%) — Large cell  3 (60%)  2(40%) 0.948 Staging (No. of patients) III B 24 (75%)  8 (25%) 0.098 IV15 (45%) 18 (55%) Chemotherapy (No. of patients) MVP 26 (67%) 13 (33%)0.199 CDDP + DOC  7 (50%)  7 (50%) 0.358 CDDP + VNR  6 (50%)  6 (50%) —Vital Capacity (% predicted) <80%  7 (78%)  2 (22%) 0.724 ≧80% 32 (57%)24 (43%) Forced Expiratory Volume in 1 second (% predicted) <70% 18(72%)  7 (28%) 0.299 ≧70% 21 (53%) 19 (47%) Use of NO Mimetics (No. ofpatients) yes 26 (81%)  6 (19%) 0.007 No 13 (39%) 20 (61%) Use ofvasodilator except for NO mimetics (No. of patients) yes 18 (69%)  8(31%) 0.246 No 21 (54%) 18 (46%)CR = complete response,PR = partial response,NC = no change,PD = progressive disease,NO = nitric oxide,MVP = mitomycin C + vinorelbine + cisplatin,CDDP = cisplatin,DOC = docetaxel,VNR = vinorelbine.Multivariate analysis was performed with Logistic regression analysis.

TABLE 3 The relationship between the use of NO donors and anticancerdrugs and chemosensitivity in a clinical research Responder;Non-responder; CR + PR NC + PD Anticancer drugs (Number) (Number) Theuse of MVP 17 26 4 6 NO donors (+) CDDP + DOC 5 1 CDDP + VNR 4 1 The useof MVP 9 13 9 20 NO donors (−) CDDP + DOC 2 6 CDDP + VNR 2 5CR = complete response,PR = partial response,NC = no change,PD = progressive disease,NO = nitric oxide,MVP = mitomycin C + vinorelbine + cisplatin,CDDP = cisplatin,DOC = docetaxel,VNR = vinorelbine.

There were no statistical significances between patients with advancedNSCLC treated with anticancer agents and NO donors and patients treatedwith anticancer agents alone in age, gender, performance status, smokinghistory, stage of lung cancer and protocol of chemotherapy (Table 1).Remarkable strong effects of the use of NO donors combined withanticancer agents on response rate to chemotherapy in patients withadvanced NSCLC were recognized as shown in Table 2 and Table 3. Responserate in patients treated with NO donors during chemotherapy (81%, 26 of32 patients) was significantly higher than that in patients treatedwithout NO donors (39%, 13 of 33 patients) (Odds ratio=6.7, 95% CI2.2-20.7, p=0.0006, Chi-square test) (Table 2). The use of NO donors(p<0.01) was significantly associated with a positive response toanticancer agents in patients with advanced NSCLC (Logistic regressionanalysis) (Table 2). Kaplan-Meier analysis showed that the survival inpatients treated with anticancer agents and NO donors was significantlylonger than that in patients treated with anticancer agents alone(p<0.05) (Log-rank test). These results (Table 2 and Table 3) suggestthat the use of NO donors, such as nitroglycerin and isosorbidedinitrate, may improve the response to chemotherapy in patients withstage IIIB or stage IV of advanced NSCLC.

Conclusions

The use of NO donors combined with anticancer agents turned out to havea great additional effect on the improvement of response rate tochemotherapy and overall survival in patients with advanced NSCLCcompared with those in patients treated with anticancer agents withoutNO donors.

Treating advanced NSCLC, squamous cell carcinoma, adenocarcinoma, andlarge cell carcinoma remains very difficult because advanced NSCLC isnot operable and the response rate to anticancer agents even if thenewest third-generation anticancer agents is very low. In contrast, theresponse rate to chemotherapy in small cell lung cancer is about 70-80%and is significantly higher than that in NSCLC. The number of patientswith lung cancer has been increasing in most countries in the world yearby year in contrast to the number of patients with gastric cancer.Furthermore, a large proportion of lung cancer consists of NSCLC.Therefore, amore effective new regimen in advanced NSCLC than currentlyexists should be established at once. On this point, the presentinvention may have a great significance on improving the response rateto not only NSCLC but other kinds of advanced solid cancer.

Example 2 Concomitant Use of a NO Donor, Nitroglycerin, ImprovesChemosensitivity in Murine Lung Cancer Model

Methods

Murine Lewis lung carcinoma (LLC) cells, lung adenocarcinoma cells, wereobtained from the Tohoku University Cell Resource Center for BiomedicalResearch and were incubated with DMEM plus 10% fetal bovine serum untilcell proliferation was sufficient to perform the experiments. The LLCcells were adjusted to concentrations of 2×10⁵ cells/100 μl withphosphate buffer saline (PBS), and were inoculated Six-week-old maleC57BL6 mice purchased from Charles River Japan, Inc. (Tokyo, Japan) andClea Japan, Inc. (Tokyo, Japan) subcutanously (2×10⁵ cells/100 μl/mouse)at the right hypochondrium. Mice were maintained underspecific-pathogen-free conditions, and provided with sterile food andwater. When transplanted tumors grew to approximately 100 mm³ in tumorvolume, animals were equally divided into four groups (Control group,n=6; C, NO donor group, n=6; N, Chemotherapy group, n=6; CTX, and NOplus chemotherapy group, n=6; N+CTX). Tumor volume was calculated as0.5×{tumor length (mm)}×{tumor width (mm)}². In the control group, 100μl of PBS was administered by intra-peritoneal injection (i.p.) fourtimes a week (once on Sunday, once on Monday, once on Wednesday, once onThursday) and 100 μl of PBS was further administered by intravenousinjection (i.v.) 30 minutes after the i.p. twice a week (once on Mondayand once on Thursday) into the C57BL6 mice. The experiments wereperformed for two weeks. In the NO donor group, 100 μl of 0.02 mg/kgnitroglycerin diluted with PBS was administered i.p. into the mice fourtimes a week (once on Sunday, once on Monday, once on Wednesday, once onThursday) and 100 μl of PBS was administered i.v. into the mice 30minutes after the nitroglycerin solution i.p. twice a week (once onMonday and once on Thursday). The experiments were performed for twoweeks. In the chemotherapy group, 100 μl of PBS was administered i.p.into the mice four times a week (once on Sunday, once on Monday, once onWednesday, once on Thursday) and 100 μl of 3.5 mg/kg cisplatin solutiondissolved in PBS was administered i.v. into the mice 30 minutes afterthe PBS i.p. twice a week (once on Monday and once on Thursday). Theexperiments were performed for two weeks. In the NO plus chemotherapygroup, 100 μl of 0.02 mg/kg nitroglycerin diluted with PBS wasadministered i.p. into the mice four times a week (once on Sunday, onceon Monday, once on Wednesday, once on Thursday) and 100 μl of 3.5 mg/kgcisplatin solution dissolved in PBS was administered i.v. into the mice30 minutes after the nitroglycerin solution i.p. twice a week (once onMonday and once on Thursday). The experiments were performed for twoweeks. Cisplatin and nitroglycerin were obtained from Nippon Kayaku Co.,Ltd (Tokyo, Japan). Measurements of murine tumor volume and mice bodyweight were performed every two days after the start of injections intomice.

Results

The tumor volume growth curve is shown in FIG. 2. Significantdiminishing of murine tumor volume was observed after day 9 in the NOplus chemotherapy group compared with that in the chemotherapy group. Onthe other hand, the NO donor group as well as the control group showedrapid tumor growth compared with that in the chemotherapy group and theNO plus chemotherapy group.

These data suggest that the chance of a new commercial use of NO donorsas an enhancing agent for effect of anticancer agents can be enlargedbecause of their excellent effect on the improvement of chemosensitivityespecially in difficult solid cancers to treat with chemotherapy.

Example 3 Concomitant Use of a NO Donor, Nitroglycerin, ImprovesChemosensitivity in Murine Colon Cancer Model

Methods

Colon 26 cells, murine colon cancer cells, were obtained from the TohokuUniversity Cell Resource Center for Biomedical Research and wereincubated with RPMI 1640 plus 10% fetal bovine serum until cellproliferation was sufficient to perform the experiments. The colon 26cells were adjusted to concentrations of 2×10⁵ cells/100 μl withphosphate buffer saline (PBS), and were inoculated Six-week-old femaleBALB/c mice purchased from Charles River Japan, Inc. (Tokyo, Japan) andClea Japan, Inc. (Tokyo, Japan) subcutanously (2×10⁵ cells/100 μl/mouse)at the right hypochondrium. Mice were maintained underspecific-pathogen-free conditions, and provided with sterile food andwater. When transplanted tumors grew to approximately 100 mm³ in tumorvolume, animals were equally divided into four groups (Control group,n=6; C, NO donor group, n=6; N, Chemotherapy group, n=6; CTX, and NOplus chemotherapy group, n=6; N+CTX). Tumor volume was calculated as0.5×{tumor length (mm)}×{tumor width(mm)}². In the control group, 100 μlof PBS was administered by intra-peritoneal injection (i.p.) four timesa week (once on Sunday, once on Monday, once on Wednesday, once onThursday) and 100 μl of PBS was further administered by intravenousinjection (i.v.) 30 minutes after the i.p. twice a week (once on Mondayand once on Thursday) into the BALB/c mice. The experiments wereperformed for two weeks. In the NO donor group, 100 μl of 0.02 mg/kgnitroglycerin diluted with PBS was administered i.p. into the mice fourtimes a week (once on Sunday, once on Monday, once on Wednesday, once onThursday) and 100 μl of PBS was administered i.v. into the mice 30minutes after the nitroglycerin solution i.p. twice a week (once onMonday and once on Thursday). The experiments were performed for twoweeks. In the chemotherapy group, 100 μl of PBS was administered i.p.into the mice four times a week (once on Sunday, once on Monday, once onWednesday, once on Thursday) and 100 μl of 3.5 mg/kg cisplatin solutiondissolved in PBS was administered i.v. into the mice 30 minutes afterthe PBS i.p. twice a week (once on Monday and once on Thursday). Theexperiments were performed for two weeks. In the NO plus chemotherapygroup, 100 μl of 0.02 mg/kg nitroglycerin diluted with PBS wasadministered i.p. into the mice four times a week (once on Sunday, onceon Monday, once on Wednesday, once on Thursday) and 100 μl of 3.5 mg/kgcisplatin solution dissolved in PBS was administered i.v. into the mice30 minutes after the nitroglycerin solution i.p. twice a week (once onMonday and once on Thursday). The experiments were performed for twoweeks. Cisplatin and nitroglycerin were obtained from Nippon Kayaku Co.,Ltd. (Tokyo, Japan). Measurements of murine tumor volume and mice bodyweight were performed every two days after the start of injections intothe mice.

Results

The tumor volume growth curve is shown in FIG. 3. Significantdiminishing of murine tumor volume was observed after day 6 in the NOplus chemotherapy group compared with that in the chemotherapy group. Onthe other hand, the NO donor group as well as the control group showedrapid tumor growth compared with that in the chemotherapy group and theNO plus chemotherapy group.

These data suggest that the use of NO donors combined with anticanceragents can enlarge the chance of a new commercial use of NO donorsbecause of their excellent effect on the improvement of chemosensitivitynot only in lung cancer but also in colon cancer.

INDUSTRIAL APPLICABILITY

The present invention is able to provide an enhancing agent for effectof anticancer agent for achieving an excellent therapeutic effect oncancer whereby it has an industrial applicability.

1. An enhancing agent for effect of anticancer agent which ischaracterized in that a nitric oxide donor is an effective ingredient.2. The enhancing agent for effect of anticancer agent according to claim1, wherein the cancer which is an object for the treatment is solidcancer.
 3. The enhancing agent for effect of anticancer agent accordingto claim 2, wherein the solid cancer is non-small cell lung cancer. 4.The enhancing agent for effect of anticancer agent according to claim 1,wherein the nitric oxide donor is an organic nitrate compound.